Erratum in

Nat Commun. 2014;5:4591.

Abstract

The auditory systems of animals that perceive sounds in air are organized to separate sound stimuli into their component frequencies. Individual tones then stimulate mechanosensory hair cells located at different positions on an elongated frequency (tonotopic) axis. During development, immature hair cells located along the axis must determine their tonotopic position in order to generate frequency-specific characteristics. Expression profiling along the developing tonotopic axis of the chick basilar papilla (BP) identified a gradient of Bmp7. Disruption of that gradient in vitro or in ovo induces changes in hair cell morphologies consistent with a loss of tonotopic organization and the formation of an organ with uniform frequency characteristics. Further, the effects of Bmp7 in determination of positional identity are shown to be mediated through activation of the Mapk, Tak1. These results indicate that graded, Bmp7-dependent, activation of Tak1 signalling controls the determination of frequency-specific hair cell characteristics along the tonotopic axis.

(a) Maximum z-projection of anE12 BP labeled with anti-Calbindin (green) and phalloidin (red)illustrating frequency-specific differences in cell morphology along the tonotopic axis. Proximal (p) and distal (d) ends are indicated. White boxes indicate regions used for zoomed images illustrated in b, b’ and b”. Top panel shows merged calbindin and phalloidin channels. White circles highlightrepresentative hair cells. Middle panel shows phalloidin-labeled stereocilia bundles highlighting the tonotopic differences in hair cell size and number. Lower panels show calbindin fluorescence in hair cells at the same BP regions. Note limited or no expression of Calbindin in hair cells (white circles) in b” . Insets: High magnification views of individual hair cell surfaces. .(c)Maximum z-projection of acontrol BP explant, labeled as in a,established at E6.5 and maintained for 6 days in vitro (IV). As for b-b”, panels illustrate changes in stereocilia size and density and Calbindin expression along the tonotopic axis.(e,f,g)Mean hair cell density, lumenal surface area and Calbindin intensity in hair cells in theindicated regions of the BP for samples maintained in ovo (black) or in vitro (grey). In all cases, a gradient is present along the tonotopic axis both in ovo and in vitro. For e, f and g, data are mean ± sem. Stars indicate a p value < 0.05 relative to the proximal value within the same group, based on Student's t-test. (h)Tonotopy is specified before E7.0. Calbindin (green) and phalloidin (red) labeling in proximal (p) and distal (d) halves from an E6.5 BP that was separated at the time of dissection and maintained for 6 days IV. The proximal-to-distal gradient ofCalbindin expression is present in the distalhalf. In contrast, Calbindin expression is almost completely absent in the proximal explant. (i)Quantification of hair cell density from specific regions (see cartoon in h) along proximal and distal explants halves. A gradient of hair cell density is present in distal explants but no change in density is present in proximal explants.Data are mean ± sem. Star indicates a p value < 0.05 relative to box 1, Student's t-test. For panel e, n=9 for both conditions, for panel f, n=8 for both conditions, For panel g, n=80 cells for each position measured from 4 separate experiments.Scale bars are 100 μm.

(a)Differential expression of members of the Bmp signalling pathway in proximal and distal regions of the BP at E6.5. Data are presented as the average PMMR (sequences per million mapped reads) forBmp7, Follistatin-like 1& 4, chordin-like 1 and Twisted gastrulation (see text for further details). All experiments were run in triplicate (n=3). (b)Expression of Bmp7 in BP tissue from the indicated regions and time points based onquantitative real-timepolymerase chain reaction (qPCR) data. An increasing proximal-to-distal gradient is present at all four time points. (c)qPCR data for Chordin-like 1 (Chdl1) indicates the presence of a counter (decreasing proximal-to-distal) gradient to Bmp7 between E6.5 and E10.(d)Twisted gastrulation, which acts to enhance the effects of Chordin-like 1, is also present in a decreasing proximal-to-distal gradient between E6.5 and E10. For b,c and d, data are mean ± sem and n=4 for each developmental age and position along the BP. (e)In situ hybridization for Bmp7at E8 (top), E10 (middle) and Chdl1(bottom)in cross-sections through the BP at the indicated locations. Both Bmp7 and Chdl1are expressed in the developing sensory epithelium of the BP (brackets) and gradients of expression are evident. A sense control for Bmp7(bottom right) was negative.

(a) Maximum z-projections from the regions indicated of BP explant cultures labeled with phalloidin. BPswere maintained for 6 daysIV from E6.5 in control media or media supplemented with0.4 μg/mL Bmp7. While hair cell density is comparable between the two conditions in the distal region of the BP, a marked increase in density is present in the proximal region of the Bmp7-treated explant. (b)Higher magnification images from the sample in a. (c)Quantification of changes in hair cell density in the indicated regions in response to Bmp7-treatment.. Treatment with Bmp7 causes a significant increase in hair cell density in the proximal region of the BP. As a result, the gradient of hair cell density across the tonotopic axis is abolished.(d)Quantification of mean hair cell lumenal surface area in control and Bmp7-treated cultures in the indicated regions of the BP. Bmp7-treatment induces a significant decrease in proximal hair cells but has no effect on distal hair cells. (e)BP explants established at E6.5, maintained for 6 DIV and then labeled with anti-Calbindin. In the Bmp7-treated explant, Calbindin expression in proximal hair cells appears comparable to expression in hair cells located in the middle region in controls. Similarly, hair cells located in the middle region in Bmp7-treated explants appear to be negative for Calbindin, a phenotype that is restricted to the distal region in control BPs. (f) Quantification of the extension of the distal calbindin phenotype (hair cells negative for Calbindin) along the tonotopic axis from the distal tip in control and Bmp7-treated explants. Bmp7 causes a significant increase in the distal extension of this phenotype. (g) Quantification of mean Calbindin intensity in hair cells from proximal and distal regions of the BP in control and Bmp7-treated explants. Bmp7-treatment causes a significant decrease in Calbindin expression in proximal hair cells.Data are presented as mean ± sem. *;p < 0.02, **; p<0.01, based on Student's t-test + Bonferroni correction. Scale bars (a = 100 μm, b = 10 μm, e = 100 μm). For ccontrols n = 14 and for Bmp7-treated n = 8.

Bmp7 treatment alters stereociliary bundle morphology in the proximal region of the BP

(a)SEM images of hair cells in the indicated regions of the BP following following 6 DIV in control or Bmp7-media. Bmp7-treatment leads to notably smaller bundles in the proximal region of the BP. (b) Quantification of the mean length of the longest stereocilia within bundles located on proximal (black bars) or distal (grey bars) hair cells. Bmp7 induces a significant increase in the mean length of the longest stereocilia of proximal hair cells. (c)Quantification of mean number of stereocilia per hair bundle in proximal (black bars) and distal (grey bars) hair cells under the specified conditions. Bmp7-treatment induces a significant decrease in the number of stereocilia per bundle in proximal hair cells. Bmp7-induced changes in hair cell stereocilia are indicated (white arrows). Data are presented as mean ± sem. **; p value < 0.05 based on Student's t-test. For controls n = 4 and for bmp7-treated n = 4.

(a) Maximal z-projections of the surface of BP explants labeled with phalloidin after 6 DIV in control media or media containing 0.4 μ/mL of Chrdl1. Note the decreased density of hair cells in the distal region in response to treatment with Chrdl1. (b) Hair cell density measured in a 2500 μm2 region at the proximal (black bars) and distal (grey bars) end of the chick BP following 6 DIV in control media, or Chrdl1-media. A significant decrease in hair cell density is observed in the distal region. (c) Lumenal surface areas for hair cells at the proximal (black bars) and distal (grey bars) ends of BP explants following 6 DIV in control media of media containing Chrdl1. Chrdl1 caused a significant increase in lumenal surface area in distal hair cells. Data are presented as mean ± sem. *; p < 0.05 based on Student's t-test. Scale bar is 50 μm. For controls n = 8, and for Chrdl1-treated cultures n = 5. Scale bar in a = 100 μm.

Over-expression of Bmp7 in ovo alters positional identity along the tonotopic axis

(a) Low magnification montage images of E14 BPs electroporated with either RCAS-AcGFP(control) orRCAS-AcGFPBmp7at E2.5. Images show BPs labeled with phalloidin (white) to visualize stereociliary bundles and anti-GFP (green) to visualize infected cells. Electroporation does not alter the normal morphology of the developing BP. (b) High magnification images of the indicated regions of the BP from control (top) and RCAS-AcGFP-Bmp7(bottom) samples labeled as in a. Expression of Bmp7 in the proximal region of the BP induces a marked increase in hair cell density and decrease in lumenal surface area. (c)Quantification of changes in mean hair cell density in proximal (black bars) and distal (grey bars) regions of the BP following electroporation with RCAS-ACFGP(control) or RCAS-AcGFP-Bmp7. Proximal Hair cell density is significantly increased in the presence of Bmp7. (d)Quantification of mean hair cell lumenal surface area following electroporation. Expression of Bmp7 induces a significant decrease in proximal hair cell surface areas.(e) Mean stereociliary bundle area for hair cells located in proximal (black bars) or distal (grey bars) regions of the BP following electroporation with either RCAS-AcGFP or RCAS-AcGFP-Bmp7.Bmp7 induces significantly decreased bundle area in proximal hair cells. Data are mean ±sem. **: p< 0.05 based on Student's t-test. Scale bars (a = 200 μm, b = 50 μm). For controls n = 4 and for Bmp7 n = 7.

Bmp7 modulates positional identity through activation of the Tak1 pathway

(a)Maximum z-projections of phalloidin-labeled BP cultures illustrating hair cell density at the proximal and distal ends of BP explants after 6 days IV under the indicated conditions (see text for details). Inhibition of Tak1 (oxozeanol) induces decreased hair cell density in the distal region of the BP, consistent with a more proximal phenotype. This phenotype contrasts with the effects of Bmp7 and is comparable to the effects of treatment with Chrdl1 (). In contrast, explants treated with both Bmp7 and oxozeanol appear comparable to control.(b) Quantification of mean hair cell density in BPs treated with oxozeanol alone or oxozeanol and Bmp7. Oxozeanol treatment leads to a significant decrease in distal density and elimination of the density gradient. In contrast, treatment with Oxozeanol and Bmp7 has no effect on the density gradient. (c,d). Data showquantification or hair cell lumenal surface area and bundle volume for the same conditions as in b. For both, Oxozeanol-treatment induces changes consistent with distalization of the BP while treatment with Oxozeanol and Bmp7 has no effect on tonotopic patterning. All data are presented as mean ± sem. Stars indicate a p value < 0.05, Student's t-test. Controls n = 14, oxozeanol n = 5, oxo + Bmp7 n = 8. (e)Western blot for total (upper) and phosphorylated (lower) Tak1 in E8 BPs in response to treatment with Bmp7. A rapid increase in levels of p-Tak1 is observed following Bmp7 treatment. Scale bar in a = 100 μm.